Collagen and Microvascularization in Placentas From Young and Older Mares

In older mares, increasing collagen fibers (fibrosis) in the endometrium and oviduct predisposes to sub-fertility and infertility. In this study, (i) gene transcription of collagen (qPCR: COL1A1, COL1A2, COL3A1, COL5A1); (ii) total collagen protein (hydroxyproline); (iii) collagen distribution (Picrosirius red staining; polarized light microscopy); and (iv) microvascular density (Periodic acid-Schiff staining), were evaluated in mares' placenta, and related to mares age, and placenta and neonate weights. Samples were collected from the gravid horn, non-gravid horn, and body of the placenta from younger (n = 7), and older mares (n = 9) of different breeds. Transcripts of COL1A1, COL3A1 and COL5A1, total collagen protein, chorionic plate connective tissue thickness, and microvascularization increased in the gravid horn of older mares' placentas, compared to the youngest (P < 0.05). Although in other species placenta fibrosis may indicate placental insufficiency and reduced neonate weight, this was not observed here. It appears that older fertile mares, with more parities, may develop a heavier, more vascularized functional placenta with more collagen, throughout a longer gestation, which enables the delivery of heavier foals. Thus, these features might represent morphological and physiological adaptations of older fertile mares' placentas to provide the appropriate nutrition to the equine fetus.

Age-Related Alterations of Hyaluronan and Collagen in Extracellular Matrix of the Muscle Spindles

Background: Muscle spindles (MSs) play a crucial role in proprioception and locomotor coordination. Although the elasticity and viscosity of the extracellular matrix (ECM) within which MSs are embedded may play a key role in MS function, the impact of aging on ECM components is unclear. The aim of the current study was to investigate the age-related physiological changes of the ECM and to verify if these could be due to alterations of the environment directly surrounding MSs. Methods: Hematoxylin Eosin and picrosirius-red staining was carried out; collagen types I (COLI) and III (COLIII) were assessed, and biotinylated hyaluronan binding protein (HABP) immunohistochemical analysis was undertaken to evaluate alterations of the ECM in the intramuscular connective tissue (IMCT) of the hindlimbs of C57BL/6J male mice. Assessments were carried out on 6-week-old (Group A), 8-month-old (Group B), and 2-year-old (Group C) laboratory mice. Results: The capsule’s outer layer became progressively thicker with aging (it was 3.02 ± 0.26 μm in Group A, 3.64 ± 0.31 μm in Group B, and 5.81 ± 0.85 μm in Group C). The collagen in IMCT around and within the MSs was significantly higher in Group C, but there were no significant differences between Groups A and B. The MS capsules and continuous IMCT were primarily made up of COLI and COLIII. The average optical density (AOD) values of COLI in IMCT surrounding MS were significantly higher after aging (p < 0.05), but there were no significant differences in COLIII in the three groups (p > 0.05). HA was present in IMCT and filled the MSs capsule. The AOD of HABP of MS showed that there were lower HA levels in Group C with respect to Group A (p = 0.022); no significant differences were noted neither between Groups A and B nor between Groups B and C (p > 0.05). Conclusion: Age-related collagen accumulation and lower HA in the ECM in which the MSs were embedded may probably cause more stiffness in the ECM in vivo, which could help to partly explain the peripheral mechanisms underlying the age-related decline in functional changes related to MSs.

Generation of a Biomimetic Substitute of the Corneal Limbus Using Decellularized Scaffolds

Patients with severe limbal damage and limbal stem cell deficiency are a therapeutic challenge. We evaluated four decellularization protocols applied to the full-thickness and half-thickness porcine limbus, and we used two cell types to recellularize the decellularized limbi. The results demonstrated that all protocols achieved efficient decellularization. However, the method that best preserved the transparency and composition of the limbus extracellular matrix was the use of 0.1% SDS applied to the half-thickness limbus. Recellularization with the limbal epithelial cell line SIRC and human adipose-derived mesenchymal stem cells (hADSCs) was able to generate a stratified epithelium able to express the limbal markers p63, pancytokeratin, and crystallin Z from day 7 in the case of SIRC and after 14–21 days of induction when hADSCs were used. Laminin and collagen IV expression was detected at the basal lamina of both cell types at days 14 and 21 of follow-up. Compared with control native limbi, tissues recellularized with SIRC showed adequate picrosirius red and alcian blue staining intensity, whereas limbi containing hADSCs showed normal collagen staining intensity. These preliminary results suggested that the limbal substitutes generated in this work share important similarities with the native limbus and could be potentially useful in the future.

Examining the Morphological and Physiological Comparisons of OVX Murine Bone vs. Mini Pigs, Rats, and Humans

Osteons are the structure and the base foundation of the human skeletal system. This cylindrical structure contains blood vessels and nerves that supply the bone matrix in humans. To study bone remodeling and bone diseases, mini pigs and rats have mainly been effective models for humans. This study was conducted to examine mouse bone structures in comparison to human, mini pig, and rat bone structures. This addresses the Three Rs principle of clinical testing on animals and proves that mice should be used as models for humans instead of mini pigs and rats. Many scientists prefer not to use mice as models for studying human bone diseases because it has been suggested that their skeletal systems are morphologically and physiologically different–as seen through aging effects. Because mice are easier to produce and can grow at a faster rate, they are more cost and time efficient to use in labs compared to rats and mini pigs. Aniline blue, Ploton silver, picrosirius red stains and TRAP and ALP cellular assays were conducted to analyze bone structures to compare with humans, mini pigs, and rats. Our data did support the hypothesis as explicit similarities between mice bone and other samples such as rats, mini pig, and human was deduced. This study concluded that there were little limitations present by using murine bone as samples for human when studying bone diseases.

Picrosirius Red Staining: Revisiting Its Application to the Qualitative and Quantitative Assessment of Collagen Type I and Type III in Tendon

Collagen has a major role in the structural organization of tendons. Picrosirius red (PSR) staining viewed under polarized light microscopy is the standard method to evaluate the organization of collagen fibers in tissues. It is also used to distinguish between type I and type III collagen in tissue sections. However, accurate analysis and interpretation of PSR images are challenging because of technical factors and historical misconceptions. The aim of this study was to clarify whether collagen types I and III can be distinguished by PSR staining in rat Achilles tendons, using double immunohistochemistry as the positive control. Our findings showed that PSR staining viewed with polarized light microscopy was suitable for qualitative and quantitative assessment of total collagen but was not able to distinguish collagen types. We found it critical to use a polarizing microscope equipped with a rotating stage; tendon section orientation at 45° with respect to crossed polarizers was optimal for the qualitative and quantitative assessment of collagen organization. Immunohistochemistry was superior to PSR staining for detection of collagen type III. We also compared formalin and Bouin solution as fixatives. Both produced similar birefringence, but formalin-fixed tendons provided higher quality histological detail with both hematoxylin–eosin and immunostaining:

Equine Sarcoid-histomorphological, Histochemical and Immunohistochemical Studies in A Thoroughbred Horse

Background: Equine sarcoid is a rare equine skin tumour seen in any age group of horses, usually in younger horses. Grossly, it appears as multinodular masses which may or may not ulcerate and pink to greyish white coloured. It was suggested that the papilloma virus is a causative organism along with predisposing factors like skin abrasions and wounds. The definitive diagnosis is based on histopathology and classified according to their gross appearance and clinical behavior. To find out the origin and proliferative nature, histochemical and immunohistochemical study was performed. The present communication reflects various patterns of sarcoid on histopathological examination and it was confirmed by histochemistry and immunohistochemistry. Methods: A four-year-old bay colt was presented to the Madras Veterinary College Teaching Hospital with nodular masses on the buccal cavity. Fine needle aspiration biopsy (FNAB), blood, serum and tumour biopsy materials were collected under local anesthesia. The collected samples were subjected to Leishman-Giemsa staining, haematological and biochemical analysis, histopathology, histochemical and immunohistochemical examination. Result: Histopathologically, there was mucosal hyperplasia and hyperkeratosis. The neoplastic cells were arranged as storiform or whorl-like pattern. Also, there was perpendicular orientation of fibroblasts towards the basement membrane (picket fence) at the dermo-epidermal junction, which was considered as characteristic feature of sarcoid. Histochemical examination with Picrosirius red revealed strong positivity characterised by deep red coloured mature collagen fibres. Immunohistochemically, the hyperplastic epithelial cells were positive for pan cytokeratin and fibroblast cells were strongly positive for vimentin. Based on the histopathology, histochemistry and immunohistochemistry, the condition was diagnosed as sarcoid which is rare in horses.

Assessment of Early Stage Glottic Cancer Depth of Resection After Transoral Laser Cordectomy

Objective Surgeons generally determine depth of resection during transoral laser cordectomy by visual inspection of the surgical field. Our aim was to examine the correlation between early glottic cancer depth of resection as reported by surgeons in the operation report and depth of resection defined by pathology specimens, using various staining techniques intended to differentiate between the distinct vocal fold layers based on particular collagen deposition. Study Design Retrospective study. Setting A voice and swallowing clinic at a tertiary referral hospital. Methods We compared depth of cordectomy assessed intraoperatively by surgeons and by pathologists using Picrosirius red stain and collagen I immunohistochemistry stain in 32 patients who underwent transoral laser cordectomy for early glottic cancer. Results For type I, II, and III cordectomy, the respective proportions of patients were 14 (47%), 9 (30%), and 7 (23%) according to surgeons’ estimations; 2 (6%), 17 (55%), and 12 (39%) according to Picrosirius red stain; and 3 (11%), 12 (44%), and 12 (45%) according to immunohistochemistry for collagen I. Conclusion Surgeons’ reported depth of resection did not correlate with depth of resection established by either staining technique. Determining depth of resection necessitates special stains, which should help in the clinical assessment of cordectomy type.

USO DE 5-FLUOROURACIL IMPACTA A PROPORÇÃO DE FIBRAS COLÁGENAS NA REGIÃO DA CABEÇA DO EPIDÍDIMO DE RATOS ADULTOS

Introdução: O uso do quimioterápico 5-fluorouracil (5-FU) pode causar toxicidade e danos colaterais a muitos órgãos. Todavia, há poucos estudos que investigam esses danos nos órgãos do sistema reprodutor masculino, por exemplo o epidídimo, o qual se destaca por armazenar e maturar os espermatozoides. Sabendo que a manutenção da histoarquitetura epididimária é importante para garantir seu desempenho funcional, faz-se relevante avaliar o colágeno presente na matriz extracelular devido ao seu papel estrutural. Objetivo: Avaliar o impacto do 5-FU nas fibras colágenas tipos I e III do epidídimo de ratos adultos. Material e métodos: Este trabalho recebeu aprovação pelo Comitê de Ética no Uso de Animais da Universidade Estadual de Maringá (protocolo: 4422140918). Quatorze ratos Wistar machos foram divididos em dois grupos (n=7): grupo controle (GC) e grupo quimioterapia (G5-FU). Os animais do G5-FU receberam, via intraperitoneal, o 5-FU por quatro dias consecutivos na dose de 15 mg/kg, com redução para 6 mg/kg por mais quatro dias alternados e dose final de 15 mg/kg. Já os animais do GC foram administrados com soro fisiológico pela mesma via e período referidos. No 15° dia, realizou-se a eutanásia dos ratos de ambos os grupos. O epidídimo foi dissecado, fixado em Methacarn e incluído em blocos de parafina para posterior realização de cortes semi-seriados de 5 μm. Estes foram corados com Picrosírius Red para realização da densitometria do colágeno na região da cabeça do epidídimo. Por fim, a análise estatística foi executada pelo teste Mann Whitney e os resultados expressos em porcentagem. Resultados: Houve diferença significativa (p<0,05) entre os grupos em relação à deposição das fibras colágenos tipo I, mas não às do tipo III. O colágeno tipo I ocupou aproximadamente 4,43% da área total no GC e 3,46% no G5-FU. Esses dados indicam que a quimioterapia impactou a organização da matriz extracelular, modificando a histoarquitetura epididimária. Mais estudos são necessários para compreender os mecanismos envolvidos bem como o reflexo disto na função do órgão. Conclusão: O 5-FU alterou a densitometria do colágeno na região da cabeça do epidídimo de ratos Wistar adultos.

Cardiac changes following arteriovenous fistula creation in a mouse model

Background: Arteriovenous fistula (AVF) creation may negatively affect cardiac structure and function and impact cardiovascular mortality. The objective of this study was to develop and characterize the cardiac changes following AVF creation in a murine AVF model. Methods: AVFs were constructed using the carotid artery and jugular vein in C57BL/6 mice. Sham-operated AVF mice served as the control group. 2D-echocardiography was performed prior to AVF creation (baseline) and at 7 and 21 days after creation in AVF and sham-operated mice. Picrosirius red was used to stain the left ventricle for collagen production. Results: The cardiac output (CO), left ventricular end diastolic (LVEDD) and systolic (LVESD) diameter, and end-diastolic (LVEDV) and systolic (LVESV) volume was significantly increased at 7 and 21 days in AVF compared to sham-operated mice. There was also a significant increase in CO, LVEDD, LVESD, LVEDV, and LVESV from baseline to 21 days within the AVF group, but not the sham-operated mice. There was a significant decrease in ejection fraction and fractional shortening at 21 days in AVF compared to sham-operated mice. Picrosirius red was significantly more prominent around both the perivascular and interstitial areas of the cardiac tissue from AVF mice compared to sham-operated AVF mice at 21 days. Conclusions: The creation of an AVF in our murine model leads to cardiac changes such as increased cardiac output, left ventricular dilation, and cardiac fibrosis, while showing reductions of ejection fraction and fractional shortening.

Automated Digital Quantification of Pulmonary Fibrosis in Human Histopathology Specimens

Pulmonary fibrosis is characterized by abnormal interstitial extracellular matrix and cellular accumulations. Methods quantifying fibrosis severity in lung histopathology samples are semi-quantitative, subjective, and analyze only portions of sections. We sought to determine whether automated computerized imaging analysis shown to continuously measure fibrosis in mice could also be applied in human samples. A pilot study was conducted to analyze a small number of specimens from patients with Hermansky-Pudlak syndrome pulmonary fibrosis (HPSPF) or idiopathic pulmonary fibrosis (IPF). Digital images of entire lung histological serial sections stained with picrosirius red and alcian blue or anti-CD68 antibody were analyzed using dedicated software to automatically quantify fibrosis, collagen, and macrophage content. Automated fibrosis quantification based on parenchymal tissue density and fibrosis score measurements was compared to pulmonary function values or Ashcroft score. Automated fibrosis quantification of HPSPF lung explants was significantly higher than that of IPF lung explants or biopsies and was also significantly higher in IPF lung explants than in IPF biopsies. A high correlation coefficient was found between some automated quantification measurements and lung function values for the three sample groups. Automated quantification of collagen content in lung sections used for digital image analyses was similar in the three groups. CD68 immunolabeled cell measurements were significantly higher in HPSPF explants than in IPF biopsies. In conclusion, computerized image analysis provides access to accurate, reader-independent pulmonary fibrosis quantification in human histopathology samples. Fibrosis, collagen content, and immunostained cells can be automatically and individually quantified from serial sections. Robust automated digital image analysis of human lung samples enhances the available tools to quantify and study fibrotic lung disease.